# 09 - 1.9 Chronobiology

# 1.9 Chronobiology

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 1.9 Chronobiology
Chronobiology is the study of biological time. The rotation of the Earth about its axis
imposes a 24-hour cyclicity on the biosphere. Although it is widely accepted that
organisms have evolved to occupy geographical niches that can be defined by the three
spatial dimensions, it is less appreciated that organisms have also evolved to occupy
temporal niches that are defined by the fourth dimension—time. Much like light
represents a small portion of the electromagnetic spectrum, the 24-hour periodicity
represents a small time domain within the spectrum of temporal biology. A broad range
of frequencies exist throughout biology, ranging from millisecond oscillations in ocular
field potentials to the 17-year cycle of emergence seen in the periodic cicada (Magicicada
spp.). Although these different periodicities all fall within the realm of chronobiology,
circadian (Latin: circa, about; dies, day) rhythms that have a period of about one day are
among the most extensively studied and best understood biological rhythms.
A defining feature of circadian rhythms is that they persist in the absence of time cues
and are not simply driven by the 24-hour environmental cycle. Experimental animals
housed for several months under constant darkness, temperature, and humidity continue
to exhibit robust circadian rhythms. Maintenance of rhythmicity in a “timeless”
environment points to the existence of an internal biological timing system that is
responsible for generating these endogenous rhythms.
The site of the primary circadian oscillator in mammals, including humans, is the
suprachiasmatic nucleus (SCN), located in the anterior hypothalamus. The mean
circadian period generated by the human SCN is approximately 24.18 hours. Like a
watch that ticks 10 minutes and 48 seconds too slowly per day, an individual with such
a period gradually comes out of synchrony with the astronomical day. In slightly more
than 3 months, a normally diurnal human would be in antiphase to the day–night cycle
and thus would become transiently nocturnal. Therefore, a circadian clock must be reset
on a regular basis to be effective at maintaining the proper phase relationships of
behavioral and physiological processes within the context of the 24-hour day.
Although factors such as temperature and humidity exhibit daily fluctuations, the
environmental parameter that most reliably corresponds to the period of Earth’s
rotation around its axis is the change in illuminance associated with the day–night cycle.
Accordingly, organisms have evolved to use this daily change in light levels as a time
cue or zeitgeber (German: zeit, time; geber, giver) to reset the endogenous circadian
clock. Regulation of the circadian pacemaker through the detection of changes in

illuminance requires a photoreceptive apparatus that communicates with the central
oscillator. This apparatus is known to reside in the eyes, because surgical removal of the
eyes renders an animal incapable of resetting its clock in response to light.
The circadian clock drives many rhythms, including rhythms in behavior, core body
temperature, sleep, feeding, drinking, and hormonal levels. One such circadianregulated hormone is the indoleamine, melatonin. Melatonin synthesis is controlled
through a multisynaptic pathway from the SCN to the pineal gland. Serum levels of
melatonin become elevated at night and return to baseline during the day. The
nocturnal rise in melatonin is a convenient marker of circadian phase. Exposure to light
elicits two distinct effects on the daily melatonin profile. First, light acutely suppresses
elevated melatonin levels, immediately decreasing them to baseline levels. Second, light
shifts the phase of the circadian rhythm of melatonin synthesis. Because melatonin can
be assayed easily, it provides a convenient window into the state of the circadian
pacemaker. Any perturbation of the clock is reflected in the melatonin profile; thus
melatonin offers an output that can be used to study the regulation of the central
circadian pacemaker.
SLEEP AND CIRCADIAN RHYTHMS
Sleep Regulation
Restful consolidated sleep is most appreciated when sleep disturbances are experienced.
Sleep is the integrated product of two oscillatory processes. The first process, frequently
referred to as the sleep homeostat, is an oscillation that stems from the accumulation and
dissipation of sleep debt. The biological substrates encoding sleep debt are not known,
although adenosine is emerging as a primary candidate neuromodulator of the sleep
homeostat. The second oscillatory process is governed by the circadian clock and
controls a daily rhythm in sleep propensity or, conversely, arousal. These interacting
oscillations can be dissociated by housing subjects in a timeless environment for several
weeks.
The circadian cycle in arousal (wakefulness) steadily increases throughout the day,
reaching a maximum immediately before the circadian increase in plasma melatonin
(Fig. 1.9-1). Arousal subsequently decreases to coincide with the circadian trough in core
body temperature. Experiments imposing forced sleep schedules throughout the
circadian day have shown that an uninterrupted 8-hour bout of sleep can only be
obtained if sleep is initiated approximately 6 hours before the temperature nadir. This
nadir typically occurs at approximately 5:00 AM to 6:00 AM. In healthy individuals,
initiating sleep between 11:00 PM and 12:00 AM affords the highest probability of getting
8 solid hours of sleep.

FIGURE 1.9-1
Relative phase relationship of sleep in young adults to other circadian phase markers.
(From Dijk D-J, Lockley SW. Invited review: Integration of human sleep-wake
regulation and circadian rhythmicity. J Appl Physiol. 2002;92:852, with permission.)
It should be stressed that diurnal preference varies among individuals as a function of
age, endogenous circadian periods, and other factors. This variability is paralleled by
physiology. Clinically, diurnal preference can be quantified using the Horne–Östberg
(HO) Morningness-Eveningness Questionnaire (MEQ). In qualitative terms, morning
people or morning larks tend to awaken earlier and experience the core body temperature
minimum at an earlier clock time relative to night people or night owls. Sleep deprivation
studies have shown that the homeostatic component of sleep is remarkably similar
among individuals of similar age. (It should be noted that there is a well-established agedependent decline in sleep need.) Therefore, diurnal preference is dictated almost
exclusively by the circadian component of sleep regulation.
Circadian Sleep Disorders
Advanced sleep phase syndrome (ASPS) is a pathological extreme of the morning lark

phenotype. An autosomal-dominant familial form of ASPS (FASPS) recently has been
genetically characterized. Afflicted family members exhibit a striking 4-hour advance of
the daily sleep–wake rhythm. They typically fall asleep at approximately 7:30 PM and
spontaneously awaken at approximately 4:30 AM. Affected individuals have a single
nucleotide polymorphism in the gene encoding hPER2, the human homolog of the mouse
Per2 clock gene. This adenine-to-guanine nucleotide polymorphism results in serine-toglycine amino acid substitution that causes the mutant protein to be inefficiently
phosphorylated by casein kinase Iε, an established component of the circadian
molecular clockwork. Similarly, delayed sleep phase syndrome (DSPS) has been shown
to be influenced by genetics. A length polymorphism in a repeat region of the hPER3
gene appears to be associated with diurnal preference in patients with DSPS, the shorter
allele being associated with evening preference.
The advent of the light bulb has extended the human day into the natural night. This
encroachment on the night, although increasing productivity, has affected human sleep
patterns (Fig. 1.9-2). Typical use of artificial lights results in a single, consolidated bout
of sleep lasting approximately 8 hours. This pattern of sleep is uncommon among most
other mammals, which typically experience more fractured sleep. Human sleep under
more natural photoperiods, where the duration of the night is longer, becomes
decompressed. Specifically, a bimodal distribution of sleep is observed; bouts of sleep
occur in early and late night. Periods of quiet wakefulness are interspersed between the
two primary bouts of sleep. This natural sleep pattern is more similar to the sleep
patterns of other mammals.

FIGURE 1.9-2
Change in sleep structure in response to artificial lighting. Total sleep time is reduced,
and periods of quiet wakefulness are abolished by extending daytime into nighttime
through artificial lighting. (From Wehr TA, Moul DE, Barbato G, et al. Conservation of
photoperiod-responsive mechanisms in humans. Am J Physiol. 1993;265:R846, with
permission.)
SEASONALITY
The 24-hour period of the Earth’s rotation around its axis is unchanging. However, the
Earth’s axis is tilted 23.45 degrees from the plane of its own orbit around the sun (the

ecliptic). As a result, the relative proportion of daytime to nighttime within the 24-hour
astronomical day varies as the Earth proceeds through its orbit of the sun. Many
organisms are capable of synchronizing physiology to the seasonal cycle to maximize
survival. For example, precise seasonal cycles in reproduction are seen throughout the
plant and animal kingdoms. Large mammals that typically have long gestation periods,
such as sheep, conceive in the fall when the nights are long and the days are short, so
birth occurs during the relatively mild season of spring. These animals are referred to as
short-day breeders. Conversely, mammals with gestation periods of only a few weeks,
such as hamsters, conceive and give birth during spring and summer, when the days are
long and the nights are short. Hence, these animals are referred to as long-day breeders.
Like circadian rhythms, many of these yearly (circannual) rhythms tend to persist in the
absence of seasonal cues with endogenous periods of approximately 1 year.
Melatonin and Seasonality
The most reliable environmental parameter providing a faithful representation of the
solar day is the day–night cycle. Similarly, the most reliable environmental parameter
reflecting the progression through the seasons is the change in day length, the fraction
of the 24-hour day between sunrise and sunset. In seasonally breeding animals, day
length is physiologically encoded through the melatonin profile. As described previously,
melatonin levels are elevated during the night. A long night, such as that experienced
during the short day lengths of winter, results in an elevated melatonin profile of a
relatively long duration. A short summer night, by contrast, results in a short duration of
elevated melatonin. This seasonal signal is interpreted by the reproductive axis,
resulting in an appropriate reproductive response. Melatonin’s role in transducing day
length was elucidated by pinealectomizing seasonally breeding animals, thereby
removing the primary endogenous source of melatonin. Melatonin was then infused in
profiles mimicking long days or short days. The duration of elevated melatonin was the
primary determinant of seasonal reproductive status, even when the infused profile was
administered under a conflicting day length. Variations in other parameters, such as the
amplitude of the melatonin profile, the amount of total melatonin synthesized, or the
phase relationship of the profile to the light–dark cycle, are of limited importance in
producing a humoral signal that transduces day length.
Reproductive responses to changing day length can be dramatic. A male Siberian
hamster (Phodopus sungorus) maintained in long days is reproductively competent and
typically has a testicular weight of approximately 250 mg per testis. Under short days,
however, the testes regress to approximately 15 mg per testis, representing a 94 percent
decrease in testicular mass. The same degree of regression is observed in response to
melatonin infusions that mimic short days. Communication of the hormonally
transduced day length to the reproductive axis is likely to be mediated, at least partially,
through melatonin receptors in the pars tuberalis of the pituitary gland. The exact
mechanism remains unknown, but activation of these receptors is hypothesized to
indirectly regulate an unidentified factor putatively named tuberalin. Tuberalin, in turn,

controls gene expression and prolactin release from lactotrophs in the adenohypophysis
of the pituitary.
Seasonality in Humans
Whether humans are truly seasonal is still a point of considerable debate. Several lines
of evidence exist that suggest the presence of a residual tendency toward seasonality. A
peak in the rate of suicide occurs in the summer; this peak is cross-cultural. Birth rates
also tend to show a seasonal variation; a small but distinguishable peak in the rate of
births occurs in spring and summer. This pattern, however, is itself variable and is
heavily influenced by unknown cultural and geographic factors. Of interest, the
amplitude of the spring–summer birth rate peak has diminished as societies have become
industrialized.
The decompressed bimodal structure of human sleep during long nights indicates that
the length of natural sleep is related to the length of the night. Potentially, a twooscillator system could function to maintain proper sleep patterns during changing
photoperiods. Such a proposed system would consist of an evening oscillator that tracks
the transition from day to night (dusk) and a morning oscillator that tracks the
transition from night to day (dawn). The relative phase differences between these
oscillators may encode the changing day lengths associated with the passing of the
seasons. Biological evidence for a two-oscillator system exists in rodents and humans.
The melatonin profile of many vertebrates, including some humans, is bimodal, with
evening and morning peaks. In rodents, metabolic and electrophysiological studies of
the SCN typically have been done in brain slices cut in the coronal plane. Results of
electrophysiological studies conducted in brain slices cut in the horizontal plane have
provided new insights. The action potential frequency in SCN neurons from horizontally
cut preparations is bimodal, with peaks in the early and late subjective day.
Furthermore, the interpeak interval varies as a function of the photoperiod in which the
animal was housed. These studies lend credence to long-standing suspicions that the SCN
of seasonally breeding mammals and, perhaps, nonseasonal mammals harbor a morning
and evening oscillator that interact to convey day-length information.
Effect of Aging
In general, as humans age, the circadian period shortens, the circadian phase advances
resulting in earlier waking times and bedtimes, the amplitudes of most circadian
rhythms decrease, and dramatic phase shifts such as those caused by jet-lag are less
tolerated. Again, a mouse model has provided interesting insight into the interaction of
the aging process and the circadian clock. The effect of chronic jet-lag on aged mice has
dramatic consequences on mortality. About half of aged mice forced to phase advance 6
hours once per week survive this treatment compared with an 83 percent survival rate
in unshifted age-matched mice. Aged mice subjected to weekly 6-hour phase delays show
an intermediate survival of 68 percent. These profound effects of phase shifting are not
observed in younger mice. The pathogenesis of chronic jet-lag remains to be determined.

Of interest, these mice did not have an increased rate of tumorigenesis. It is likely that
in humans, as in mice, the internal desynchrony of oscillators that result from a rotating
light schedule may have dire consequences that may be exacerbated by aging.
CIRCADIAN RHYTHMS AND PHARMACOTHERAPY
Circadian rhythmicity can be affected by drugs, and conversely, the circadian clock can
modulate the efficacy of drugs throughout the course of the day. A better understanding
of these interactions will lead to more effective pharmacotherapies. Some of the beststudied interactions between medications and the circadian clock have included the
circadian effects of antidepressants. Elevated nocturnal body temperature is a common
feature among depressed patients. This effect may be due to a reduced amplitude of the
master circadian oscillator in the hypothalamus that drives body temperature. Tricyclic
antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs) reduce
elevated nocturnal body temperature while simultaneously enhancing circadian
amplitude. Similarly, many depressed patients exhibit a dampened amplitude in daily
activity rhythms. Like body temperature, the amplitude in daily activity cycles of
depressed individuals may be augmented by TCA or SSRI therapy.
The use of lithium to treat bipolar disorder has been long established. However,
lithium also affects the circadian system, resulting in a lengthening of circadian period.
The molecular mechanism by which this occurs remains unknown. Glycogen synthase
kinase 3β (GSK3β) has been implicated in participating within the molecular clock
mechanism. Of interest, GSK3β is inhibited by lithium. In cell culture, GSK3β has been
shown to stabilize the negative clockwork regulator REV-ERBα via phosphorylation.
REV-ERBα typically represses the transcription of the BMAL1 gene. In the presence of
lithium, however, GSK3β is inhibited, thereby preventing the phosphorylation and
stabilization of REV-ERBα, which as a consequence is targeted for proteasomal
degradation. The degradation of REV-ERBα results in the de-repression of BMAL1
transcription. Whether lithium’s influence on circadian behavior is attributable to its
inhibitory effect on GSK3β-mediated stabilization of REV-ERBα remains to be
determined.
Short-acting benzodiazepines (e.g., triazolam [Halcion] and brotizolam [Lendormin])
also exert chronobiological effects. In hamsters, triazolam or brotizolam administered
during the middle of the subjective day induces circadian phase advances in activity.
Brotizolam has been shown to reduce the light-induced expression of clock genes Per1
and Per2 in the SCN. Although benzodiazepines are allosteric modulators of γaminobutyric acid A receptors (GABAA), several lines of evidence indicate that the
circadian effects of short-acting benzodiazepines require an intact serotonergic system.
When the 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT) is injected into hamsters at subjective midday, phase advances in locomotor
behavior and SCN neuronal activity are observed in addition to a reduction in Per1 and
Per2 gene expression in the SCN. Recreational drugs of abuse also affect the circadian
system. 3,4-Methylenedioxymethamphetamine (MDMA), or “ecstasy,” can act as a

serotonin neurotoxin. Hamsters treated with MDMA showed reduced triazolam-induced
phase shifts in circadian locomotor activity and a diminished ability to reentrain
rhythms posttreatment. MDMA-treated animals exhibited a reduction of serotonergic
axonal terminals in the SCN, again emphasizing the importance of an intact
serotonergic system in the regulation of the circadian axis. Recreational use of
methamphetamine 
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increased 
dramatically. 
Chronic 
administration 
of
methamphetamine disorganizes rodent activity rhythms. However, administration of
methamphetamine to rodents rendered arrhythmic through ablation of the SCN results in
a reemergence of rhythmicity. The mechanism involved in the rescue of rhythmicity or
site of action remains unknown.
The efficacy and toxicity of many pharmacotherapeutics vary as a function of
circadian phase. Daily variations in fixed-dose lethal toxicity have been appreciated in
rodents for years. Many anticancer drugs, ranging in mechanism from antimetabolites
to deoxyribonucleic acid (DNA) intercalators to mitotic inhibitors, have been shown to
have 2- to 10-fold changes in tolerability in rodents over the course of the day. Much of
this difference is attributed to circadian variations in the body’s ability to absorb,
distribute, metabolize, and eliminate toxic compounds. These four processes are affected
by underlying circadian rhythms in physiological processes such as daily variations in
gastric pH, gastrointestinal mobility, glomerular filtration rate, and membrane
viscosity. The rhythmic intake of food during traditionally timed meals also influences
how therapeutic drugs are handled by the body. It is becoming clear that to maximize
efficacy and minimize toxicity of drugs, the circadian phase of administration must be
considered. Appropriate circadian timing of the administration of multiple drugs can be
a daunting challenge to infirmed individuals or their caretakers. The development of
small implanted programmable pumps that can be directed to administer anticancer
drugs or other therapeutics at particular times of day may provide a limited solution to
this challenge. The emergence of the field of chronotherapy is a reflection of our
increased understanding of the impact of the circadian system on the effectiveness of
pharmacological treatments.
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